Limitations and Possibilities of the System

The main limitations of the translocation system described here are that (a) many passenger proteins interfere with the structure of the toxin so that it cannot recognize the toxin receptor, and that (b) the passenger protein may not be able to unfold. We have tested a large number of proteins, and most of them are not translocated due to one of these problems (Klingenberg and Olsnes, 1996). This may also be a problem with smaller peptides, but usually less than with protein passengers. Conceivably, modification of the fusion proteins with polypeptide linkers and by mutations in the passenger protein may overcome the problem, but this will require a considerable amount of work in each case. 

The question whether toxins can be used to translocate nucleic acids into cells is very interesting, but this has not been tested seriously up to now. Toxins are interesting candidates for delivery of anti-sense RNA, ribozymes and genes for transformation and gene therapy. So far there is not convincing evidence that toxins have been effective for such purposes. From what is now known about the translocation of diphtheria toxin, it is likely that the nucleic acid would have to be linked end-to-end to the N-terminus of the A-fragment of this 

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